This proposal focuses on endocytic processes that remove transporters, specifically cardiac Na/Ca exchangers (NCX1), from the surface membrane. Membrane fusion and budding processes are fundamental to all eukaryotic life, and we have developed improved electrophysiological methods to analyze trafficking events at the cell surface, starting with immortalized fibroblasts and proceeding to adult cardiac myocytes. Exploiting unprecedented control of the cytoplasmic milieu with high resolution capacitance recording, we have discovered that cytoplasmic ATP depletion, followed by a Ca transient and ATP replenishment, promotes a massive endocytic response (MEND). We have further determined that NCX1 is internalized during MEND. As NCX1 plays a major role in ischemia-reperfusion damage and related cardiac arrhythmias, removal of NCX1 from the membrane in response to metabolic stress can be of substantial clinical significance. Therefore, we have initiated a detailed analysis of the MEND response. Preliminary Data indicates that MEND is driven by remodeling of actin membrane cytoskeleton with ATP-, Ca- and PIP2- dependent processes all playing essential roles. Further Preliminary Data shows that NCX1 lateral mobility decreases dramatically in steps leading up to MEND, as well as with stabilization of F-actin. Therefore, we will analyze how metabolic state regulates actin cytoskeleton and NCX1-actin cytoskeleton interactions. Additionally, we will identify the Ca sensors underlying MEND, and we will analyze how PIP-kinases involved in MEND are regulated. To address how NCX1 couples to MEND, new NCX1 fusion proteins have been developed for on-line monitoring of NCX1 internalization, pulse-chase tracking of NCX1, and improved analysis of NCX1 mobility. An NCX1 fusion with Dendra2 allows conversion of green transporters to red transporters, followed by tracking of the two transporter species. Halotag fusions on the extracellular side allow sequential NCX1 labeling with different membrane-permeable and -impermeable fluorophores. In the longer term, these fusions will allow the use of quantum dots and Nanogold to study NCX1 trafficking. Overall, the proposed work will generate fundamental insights into a powerful endocytic process that is of wide cell biological interest and is likely to play an important role in cardiac ischemia-reperfusion and related pathologies. PUBLIC HEALTH RELEVANCE: Public Health Relevance Cardiovascular disease is the leading cause of death in the United States. Many deaths in the immediate aftermath of myocardial infarction are caused by cardiac arrhythmias, and in the long-term of cardiac insufficiency malfunction of cardiac excitation-contraction coupling and associated arrhythmias are thought to play an important role. The pathogenesis of arrhythmias is complex and involves numerous molecular entities. The cardiac Na/Ca exchanger, which removes Ca from cardiac myocytes and is the major focus of this study, is thought to play a trigger role in many cases by generating inward membrane current. Also, this transporter is implicated to mediate much cardiac cell damage from ischemia-reperfusion episodes by loading cardiac cells with calcium in response to previous Na loading, thereby causing myocyte hypercontraction and promoting cell death programs to be activated via mitochondrial signaling mechanisms that are set in motion. The experimental program addresses how Na/Ca exchangers may be removed from the cell surface membrane and how this process may be regulated, in particular how it may become inactivated in pathological settings. Endocytic mechanisms have been found to be activated in multiple non-cardiac cell types in response to ischemia and/or oxygen deprivation. We will now explore related mechanisms in cardiac myocytes. To do so, we are taking a highly unique approach by starting from analysis of endocytic mechanisms and their regulation in simple cell culture cells, where Na/Ca exchangers can be expressed, and proceeding to the analysis of the equivalent mechanisms in cardiac myocytes. Our overall goal is a better understanding of the `life-time'and endocytosis of NCX1. This work can be expected to have fundamental implications for cardiac pathologies and ultimately medicine.